Project Summary/Abstract One of the goals of our TRiC Chaperonin Network PPG is to understand the basis of Huntington's disease pathogenesis by studying the structure of disease-causing proteins in oligomers and larger protein aggregation assemblies together with investigating the cellular machinery needed for neuronal function and survival. Using these mechanistic insights, we can evaluate the effect of TRiC chaperonin reagents on modulating protein and cellular dysregulation. Cryo-ET was used to investigate cellular structures in HD iPSC-derived i-neurons that might be altered by mHTT. A control line (with 18Q) and an expanded repeat HTT line (77Q) were differentiated to mature neurons with medium spiny striatal characteristics and methods to enable differentiation on the electron microscope (EM) grids under conditions compatible for cryo-ET were employed. The Thompson and Chiu labs developed a protocol for the first time whereby iPSC-neurons were grown on EM grids. Frozen grids were then shipped to Stanford overnight and examined in the cryo-EM facility. HD iPSC- neurons in a frozen, hydrated state without fixative or stain show remarkably dense features in mitochondria. Such features are not seen with normal repeat (18Q) iPSC-neurons prepared under identical conditions. These studies show that cryo-ET can be used to identify structural differences in iPSC-neurons expressing disease- causing mHTT protein species. In very new data, we now can observe dense protein ?hairballs? in the neurites of two different HD iPSC-derived neurons. We propose to use this innovative and novel technological advance to carry out studies using HD and control iPSC neurons with cryo-ET and functional studies to determine protein species and cellular organelle morphologies that may predict disease toxicity. One of the hurdles to this approach has been the lack of a vitrification device in the Thompson lab, which is responsible for growth and differentiation of the iPSCs on the EM grids, followed by the freezing step prior to sending to Stanford. To date, research staff from Stanford has come down to UCI to work with the Thompson lab and use a vitrification device housed in the EM core facility at UCI, which is located across campus. However, while this has provided extremely promising results that are consistent with HD pathogenesis, we are cautious of our observed features, which could be induced by ?motion stress? of the cells on the EM grids, due to the transport across campus for vitrification. Though we do not see such features in the control cells, we cannot rule out that the high Q iPSC lines may be more susceptible to ?motion stress?. This concern was based on our previous observations of stress granules observed in cryo-ET of neurons before Dr. Chiu's lab was equipped with cell culture lab adjacent to the vitrification device. Therefore, we propose to seek supplement funds to purchase a vitrification device as used in the Chiu lab and to place it right next to the tissue culture lab in the Thompson lab. The personnel in the Thompson lab will be trained by the Chiu staff to perform the plunge freezing step. We therefore propose the purchase of such a device with this administrative supplement.